Human cytomegalovirus (HCMV) causes one of the most common opportunistic infections encountered in patients with AIDS. Although the only two available drugs, ganciclovir and foscarnet, are effective for HCMV therapy, the emergence of drug-resistant HCMV strains has posed a need to develop new drugs and novel strategies to combat HCMV infections. The goal of the proposed research is to develop small novel RNA molecules as anti-HCMV therapeutic agents by directing endogenous RNase P to hydrolyze a HCMV essential mRNA and inhibit viral gene expression and replication. These RNA molecules, termed as external guide sequences (EGS), consist of a sequence antisense to the target mRNA and a sequence that guides RNase P to cleave the target. Recent studies in our and other laboratories have shown that custom-designed EGSs can target both cellular and viral mRNAs for RNase P degradation and inhibit their expression in tissue culture. In this research program, we will determine whether EGSs can be generated to direct human RNase P to cleave the overlapping region (AP mRNA) of the mRNAs encoding the HCMV capsid assembly protein (AP) and protease (PR) and weather these EGSs can inhibit viral replication by abolishing the AP and PR expression. Initially, highly active and sequence-specific EGSs that target AP mRNA will be constructed. Second, how EGSs direct RNase P to cleave the AP mRNA will be studied by both in vitro kinetic and structural analyses. Third, the requirements for efficient EGS activity in tissue culture will be defined and the sequence specificity determined. Finally, whether EGSs can inhibit HCMV replication by abolishing AP and PR expression will be investigated. These studies will reveal the mechanism of how EGSs achieve high targeting efficacy and sequence specificity and facilitate the development of these novel RNA agents for antiviral and anticancer applications.